GABAergic neurons of anterior thalamic reticular nucleus regulate states of consciousness in propofol‐ and isoflurane‐mediated general anesthesia

Abstract Background The thalamus system plays critical roles in the regulation of reversible unconsciousness induced by general anesthetics, especially the arousal stage of general anesthesia (GA). But the function of thalamus in GA‐induced loss of consciousness (LOC) is little known. The thalamic reticular nucleus (TRN) is the only GABAergic neurons‐composed nucleus in the thalamus, which is composed of parvalbumin (PV) and somatostatin (SST)‐expressing GABAergic neurons. The anterior sector of TRN (aTRN) is indicated to participate in the induction of anesthesia, but the roles remain unclear. This study aimed to reveal the role of the aTRN in propofol and isoflurane anesthesia. Methods We first set up c‐Fos straining to monitor the activity variation of aTRNPV and aTRNSST neurons during propofol and isoflurane anesthesia. Subsequently, optogenetic tools were utilized to activate aTRNPV and aTRNSST neurons to elucidate the roles of aTRNPV and aTRNSST neurons in propofol and isoflurane anesthesia. Electroencephalogram (EEG) recordings and behavioral tests were recorded and analyzed. Lastly, chemogenetic activation of the aTRNPV neurons was applied to confirm the function of the aTRN neurons in propofol and isoflurane anesthesia. Results c‐Fos straining showed that both aTRNPV and aTRNSST neurons are activated during the LOC period of propofol and isoflurane anesthesia. Optogenetic activation of aTRNPV and aTRNSST neurons promoted isoflurane induction and delayed the recovery of consciousness (ROC) after propofol and isoflurane anesthesia, meanwhile chemogenetic activation of the aTRNPV neurons displayed the similar effects. Moreover, optogenetic and chemogenetic activation of the aTRN neurons resulted in the accumulated burst suppression ratio (BSR) during propofol and isoflurane GA, although they represented different effects on the power distribution of EEG frequency. Conclusion Our findings reveal that the aTRN GABAergic neurons play a critical role in promoting the induction of propofol‐ and isoflurane‐mediated GA.


| INTRODUC TI ON
2][3] However, the neural mechanism of GA-induced loss and recovery of consciousness remains unclear.
0][11][12][13] Moreover, multiple nuclei in the thalamus, such as the paraventricular thalamus (PVT), the central medial thalamic nucleus (CMT) and their related neural circuits, have been reported to play important roles in the regulation of states of consciousness during GA, especially the recovery period after GA. [14][15][16][17][18][19][20][21] A recent study also revealed that the ventral posteromedial nucleus (VPM) of the thalamus actively promoted arousal of GA, which is independent of the process of anesthesia. 22These findings suggested the importance of the thalamus in the recovery of consciousness after GA.However, the roles and mechanisms of the thalamus in GA-induced consciousness loss were barely known.
The thalamic reticular nucleus (TRN) is the only GABAergic neurons-composed nucleus in the thalamus, which is the main source of inhibition for the thalamus. 23,246][27][28][29][30] The TRN was reported to synchronize brainwide network activity. 25,26,31Brief optogenetic stimulation of TRN neurons induces spindles in the cortex, 32 whereas the alpha oscillations (8-12 Hz) in the cortex is identified as a characteristic of propofol-induced anesthesia, [33][34][35][36][37] which indicated the involvement of TRN in GA regulation.Furthermore, manipulation of the basal forebrain (BF)-TRN or the locus coeruleus (LC)-TRN pathway affected the progress of isoflurane-or propofol-mediated GA. 38,39 The TRN is anatomically divided into several sectors distributed along the plane of the nucleus, and each sector forms distinct neural circuits which are composed of the specific cortical afferents and the thalamic target of TRN neurons. 40The anterior sector of the TRN (aTRN) receives the projections from the prefrontal cortex (PFC) 41 and sends mainly outputs to the thalamic nonspecific nuclei, such as the thalamic mediodorsal nucleus (MD) and the VPM 40 to play a part the regulation of the corticothalamic and thalamocortical activities. 41These findings suggested that the aTRN neurons may participate in GA-induced loss and recovery of consciousness, but the roles remain unclear.
In the present study, we investigated how GABAergic neurons in aTRN regulate GA induced by propofol and isoflurane.c-Fos staining was set up to monitor the activity variation of aTRN neurons during distinct stages of GA.Subsequently, optogenetic or chemogenetic activation of aTRN GABAergic neurons was performed to clarify the function of aTRN during propofol-/isoflurane-mediated GA.
Taken together, our findings show a critical role of aTRN GABAergic neurons in the modulation of propofol-and isoflurane-induced unconsciousness.

| Stereotaxic surgery
Mice were placed on a stereotaxic apparatus (RWD Life Science, Shenzhen, China) and anesthetized with 1.4% isoflurane.For local analgesia, lidocaine was subcutaneously injected before the skull surface exposure.

| Behavioral tests
The induction time of loss of righting reflex (LORR) and the duration time recovery of righting reflex (RORR) are used as standard indices of GA induction and emergence in mice, respectively.
For propofol anesthesia, a single dose of 20 mg/kg propofol was administered through the caudal vein.The duration of anesthesia was defined as the RORR time.For isoflurane anesthesia, mice were placed in a recording chamber (RWD Company, Shenzhen, China) and incubated with 1.4% isoflurane in 100% oxygen at a rate of 1 L/ min.An anesthesia monitor (Vamos; Drager Company, Germany) was connected to detect the concentration of isoflurane in the anesthesia chamber and an electric blanket with a rectal temperature probe was used to the bottom of the anesthesia chamber and was controlled at 37.5°C in the whole experiment.The interval between the starting point of isoflurane application and the time point at which mice lost the righting reflex was regarded as the latency to LORR.The mice were kept anesthetized for 20 min.After that, we turn off isoflurane infusion and immediately exhaust the remaining isoflurane in the chamber.The duration from the end of isoflurane infusion to RORR of mice was defined as RORR time.There was at least 7 days rest between light-on and light-off or CNO and saline treatment and isoflurane or propofol administration in the same mouse.
For optogenetic experiments, blue laser (473 nm, 5 mw, 10 Hz, 10 ms) stimulation was delivered to the mice during induction and emergence of GA.For induction, opto-stimulation was administered with isoflurane inhalation and continued until the mice achieved LORR.For emergence, the mice were optically stimulated from the cessation of isoflurane/propofol application until RORR.
In the lesion experiments, we recorded the LORR under isoflurane anesthesia and RORR under propofol/isoflurane anesthesia.
Immunofluorescence analysis was performed on all mice to verify the position and efficiency of viral transfection after all tests were completed.

| EEG recording
EEG was recorded using a multichannel signal acquisition system (Appolo, Bio-Signal Technologies, USA) and then digitized and analyzed using the Spike2 software (Cambridge Electronic Design, Cambridge, UK).EEG signals were recorded for 5 min before induction and were continuously recorded until recovery from propofol or isoflurane anesthesia.Relative powers in the different frequency bands were computed by averaging the signal power across the frequency range of each band (delta (δ): 1-4 Hz, theta (θ): 4-8 Hz, alpha (α): 8-12 Hz, beta (β): 12-25 Hz, and gamma (γ): 25-60 Hz) and then dividing by the total power from 1 to 60 Hz.MATLAB (R2020a; MathWorks, Natick, MA, USA) and customized MATLAB codes were used for burst suppression ratio (BSR) calculation. 42We calculated the BSR in the LORR and RORR period of isoflurane anesthesia and the RORR period of propofol anesthesia in the optogenetic experiments.In the chemogenetic experiments, we calculated the BSR from the administration of isoflurane/propofol to the RORR of the mice.

| In vitro electrophysiological recording
Three weeks after virus transfection, the brains of PV-IRES-cre or SST-IRES-cre mice were dissected out and incubated in oxygenated

| Immunohistochemistry
Mouse brains were collected after behavioral tests and EEG recordings.The brain tissues were fixed in 4% PFA and dehydrated in 30% sucrose, then frozen in OCT and sectioned at 20 μm using a freezing microtome (Leica CM1900, Germany).Sections containing aTRN were blocked in blocking buffer (10% goat serum, 1% BSA, and 0.3% Triton in PBS) for 2 h at room temperature (RT), followed by incubating with primary antibodies overnight at 4°C and secondary antibodies for 2 h at RT.The primary antibodies used were 1:1000 for c-Fos, 1:200 for SST, and 1:1000 for PV.All secondary antibodies were diluted to 1:500.Sections were mounted using the DAPI Fluoromount-G (Southern Biotech).All images were captured by a virtual microscopy system (Olympus BX63) and a laser confocal fluorescence microscope (Olympus FV1000).Only data from mice in which the AAV infection and the location of the optical fiber were confirmed were included.In this study, mice with poor AAV expression, misplaced optical fibers, or loose electrodes were excluded from further analysis.

| Statistical analysis
We utilized GraphPadPrism 8.0 (GraphPad Software Inc., USA) for statistical analyses.All data were subject to tests for normality.The cell counting of c-Fos staining among the wake, propofol/ isoflurane and recovery groups was analyzed by one-way ANOVA.
IBO experiments were detected using the independent-samples t-tests.Independent-samples t-test was also applied in the analysis of LORR and RORR times between the control and IBO groups.

| aTRN GABAergic neurons are activated during the induction of GA
It is reported that TRN is composed of GABAergic neurons, which nearly all express PV and partly express SST. 43PV/SST double staining was set up to confirm the composition of aTRN.The coexpression proportion of PV and SST neurons in TRN is about 40.5%, and the proportion of independent expression of PV or SST neurons is about 38% and 21.5%, respectively (Figure S1A,B).Furthermore, VGAT-tdTomato (VGAT-td) mice were used to monitor the proportion of PV or SST neurons in aTRN and we found that the proportion of PV is about 80.6%, whereas the proportion of SST is 56.8% (Figure S1C,D).Our data showed that about 40% of aTRN neurons are PV + /SST + double positive, which is different from the separated   A single dose (20 mg/kg) of propofol was used to reveal the effect of aTRN PV neuron activation on propofol-induced anesthesia.The tail vein injection of 20 mg/kg propofol caused an immediate coma in mice; therefore, we can evaluate the effect of aTRN PV neurons on the emergence of propofol anesthesia (RORR).

| Optogenetic activation of aTRN PV Neurons delays the emergence of propofol anesthesia
Optogenetic activation of aTRN PV neurons prolonged the RORR time of propofol anesthesia (Figure 2D).EEG recordings were set up to further assess how aTRN PV neurons affect propofol anesthesia.In the ChR2-expressing mice, the burst suppression ratio (BSR) was notably increased during propofol anesthesia after aTRN PV neuron activation (Figure 2E), which indicated the deeper anesthesia. 42

| Optogenetic activation of aTRN PV Neurons promotes the induction and delays the emergence of isoflurane anesthesia
We further tested the effect of aTRN PV neuron activation on isoflurane anesthesia (Figure 3A).Activation of aTRN PV neurons by blue laser stimulation shortened the induction time (LORR) and prolonged the emergence time (RORR) of isoflurane anesthesia (Figure 3B,C).In the ChR2-expressing mice, activation of aTRN PV neurons increased the BSR in the emergence period of isoflurane anesthesia (Figure 3D), which is consistent with the propofol anesthesia (Figure 2E).EEG recordings also showed decreased δ wave (1-4 Hz) proportion and increased α wave (8-12 Hz) proportion during the induction of isoflurane anesthesia in the ChR2expressing mice after blue laser stimulation (Figure 3E,G Taken together, our data indicate that optogenetic activation of aTRN PV neurons promotes the induction of isoflurane anesthesia and delays the recovery of both propofol and isoflurane anesthesia.

| Optogenetic activation of aTRN SST Neurons decelerates the emergence of propofol anesthesia
Next, we generated ChR2-transfected mice to access the roles of aTRN SST neurons in propofol anesthesia.rAAV-EF1α-DIO-hChR2-mCherry virus (ChR2) was unilaterally injected into aTRN of the SST-IRES-cre mice, followed placement of an optical fiber in the same position (Figures 2A and 4A, Figure S3A).Electrophysiological recording in the ex vivo brain slices confirmed the activation of aTRN SST neurons (Figure 4B).
After 20 mg/kg propofol injection into the mice, the effect of optogenetic activation of aTRN SST neurons on RORR, and the EEG signature of propofol anesthesia was detected.We found that optogenetic activation of aTRN SST neurons prolonged the RORR time of propofol anesthesia (Figure 4C).EEG recordings showed that the BSR was increased during propofol anesthesia after the activation of aTRN SST neurons (Figure 4D).Furthermore, similar to aTRN PV neurons, optogenetic activation of aTRN SST neurons caused a decreased δ wave (1-4 Hz) percentage and an increased percentage of α wave

| Optogenetic activation of aTRN SST Neurons accelerates the induction and decelerates the emergence of isoflurane anesthesia
Furthermore, we tested whether activation of aTRN SST neurons affects isoflurane anesthesia (Figure 3A).We found that activation of aTRN SST neurons by blue laser accelerated the LORR time and delayed the RORR time of isoflurane anesthesia (Figure 5A,B).Similar to activation of aTRN PV neurons, activation of aTRN SST neurons also increased the BSR in the emergence period of isoflurane anesthesia (Figure 5C).EEG recordings showed decreased δ wave (1-4 Hz) percentage and increased percentage of α wave (8-12 Hz) and γ wave (25-60 Hz) during both the LORR induction and the emergence period of isoflurane anesthesia in the ChR2-expressing mice after blue laser stimulation (Figure 5D-G, Figure S3D-G).
Combining the data from optogenetic activation of aTRN PV and aTRN SST neurons, we draw the conclusion that both aTRN PV and aTRN SST neurons promotes the induction of isoflurane anesthesia and delays the recovery of both propofol and isoflurane anesthesia.But the EEG recordings showed that optogenetic activation of aTRN PV and aTRN SST neurons both decreased δ wave (1-4 Hz) proportion, which referred the cortical wakefulness. 44This is inconsistent with the promotion effect for anesthesia of these two types of aTRN neurons.To clarify the precise functions of aTRN PV and aTRN SST neurons in anesthesia, chemogenetic tools for continuous manipulation of aTRN neurons were generated for behavior tests and EEG recording.

| Chemogenetic activation of aTRN PV Neurons delays the emergence of propofol anesthesia
Previous studies and our data revealed that aTRN is mainly composed of PV + GABAergic neurons, 45 and we found shared roles of aTRN PV and aTRN SST neurons in anesthesia.So we used PV-IRES-cre mice to study the effect of chemogenetic activation of aTRN neurons on propofol and isoflurane anesthesia.To continuously modulate the activity of aTRN PV neurons, rAAV-EF1α-DIO-hM3Dq-EGFP (M3) virus was unilaterally microinjected into aTRN of the PV-IRES-cre mice (Figure 6A).Immunofluorescence staining confirmed virus expression in the aTRN PV neurons (Figure 6B and Figure S4A).One hour before propofol application, CNO was intraperitoneally injected to activate the aTRN PV neurons.The electrophysiology recording on the brain slice showed increased firing of aTRN PV neurons after CNO injection in the M3-expressing mice (Figure 6C).Activation of aTRN PV neurons by CNO significantly prolonged the emergence time (RORR) after 20 mg/kg propofol injection (Figure 6D).In the M3-expressing mice, activation of aTRN PV neurons by CNO also increased the BSR during the RORR period of propofol anesthesia (Figure 6E).On the contrary of optogenetic activation of aTRN PV neurons, TRN PV neuron activation by CNO increased the percentage of the δ wave (1-4 Hz) and decreased the γ wave (25-60 Hz) proportion during the RORR period of propofol anesthesia (Figure 6F-H, Figure S4B,C), which is consistent with the delay effect for emergence of propofol anesthesia.

| Chemogenetic activation of aTRN PV neurons promotes the induction and delays the emergence of isoflurane anesthesia
During isoflurane anesthesia, shortened LORR time and prolonged RORR time were observed in the CNO treated M3-expressing mice compared to the M3-saline and EGFP-CNO groups (Figure 7A-C).
In the M3-expressing mice, activation of aTRN PV neurons by CNO administration significantly increased the BSR during the maintenance and emergence period of isoflurane anesthesia (Figure 7D).Combining the data from optogenetic and chemogenetic activation of aTRN neurons, we conclude that activation of aTRN GABAergic neurons promotes the induction of isoflurane anesthesia and delays the recovery of propofol/isoflurane anesthesia.

| Manipulation of aTRN VGAT neurons regulates the behavior of propofol and isoflurane anesthesia
Then we used VGAT-IRES-cre mice to study the effect of the whole mount aTRN GABAergic neurons manipulation on propofol and isoflurane anesthesia.rAAV-EF1α-DIO-hChR2-EGFP virus (ChR2) was unilaterally injected into aTRN of the VGAT-IRES-cre mice, followed placement of an optical fiber in the same position (Figure 8A).Optogenetic activation of aTRN VGAT neurons prolonged the RORR time of propofol anesthesia (Figure 8B).Accelerated the LORR time and delayed the RORR time were also observed after activation of aTRN VGAT neurons during isoflurane anesthesia (Figure 8C).Next, we performed the chemogenetic inhibition strategy to confirm the role of aTRN VGAT neurons in GA. rAAV-EF1α-DIO-hM4Di-EGFP (M4) virus was bilaterally microinjected into aTRN of the VGAT-IRES-cre mice (Figure 8D).One hour before propofol application, CNO was intraperitoneally injected to deactivate the aTRN VGAT neurons.
Interestingly, we only found the prolonged LORR time of isoflurane anesthesia (Figure 8F), the RORR time of propofol or isoflurane anesthesia was not significantly altered (Figure 8E,F).At last, to test which part of TRN regulates anesthesia progression, we injected IBO into aTRN or the posterior sector of the TRN (pTRN) of the VGAT-td mice to kill aTRN or pTRN neurons, respectively.Seven days after IBO injection, the number of tdTomatolabeled aTRN or pTRN neurons was massively decreased in the lesion group (Figure S5A,B,E,F).We found a shorter RORR time in the aTRN lesion mice in propofol anesthesia (Figure S5C).Longer LORR time and shorter RORR time were observed in the aTRN lesion mice during isoflurane anesthesia (Figure S5D).On the contrary, pTRN lesion did not alter the behaviors of propofol or isoflurane anesthesia (Figures S5G,H).These data suggest that aTRN may be the essential region of TRN for the regulation of GA.

| DISCUSS ION
In this study, we set up immunofluorescence, optogenetic, chemogenetic and EEG recording techniques to investigate the roles of the aTRN GABAergic neurons in regulating the process of propofol/ isoflurane anesthesia.PV/SST double staining showed that aTRN is composed of PV and SST subtypes of GABAergic neurons.c-Fos imaging showed that the activity of both aTRN PV and aTRN SST neurons was increased during propofol/isoflurane induced anesthesia and decreased during recovery period after anesthesia.Optogenetic activation of both aTRN PV and aTRN SST neurons promoted the induction of isoflurane anesthesia and delayed the emergence of both propofol and isoflurane anesthesia.Accordingly, Optogenetic activation of aTRN PV and aTRN SST neurons accumulated the BSR during propofol/isoflurane induced anesthesia.Additionally, chemogenetic activation of aTRN PV neurons also accelerated the induction time of isoflurane anesthesia and prolonged the emergence time of propofol/isoflurane anesthesia and increased the BSR during propofol/isoflurane induced anesthesia.Furthermore, the power distribution of EEG frequency is different under optogenetic and chemogenetic activation of aTRN neurons, respectively, which may attribute to the specific role of aTRN neurons on cortical spindles.Taken together, these findings suggest a critical role of aTRN in controlling the process of propofol-and isoflurane-mediated anesthesia.
The TRN is the only GABAergic neurons formed neural nucleus in the thalamus, which is the main inhibition source for the thalamus. 23,24The TRN was reported to include almost PV-expressing GABAergic neurons, which was considered as a homogeneous GABAergic neuron formed nucleus. 46,47But SST-expressing neurons were identified in TRN recently, 45,48 and TRN neurons have been reported to exhibit heterogeneities in molecular and electrophysiological properties, and neural connectivity. 28,43,45,48,49These finding of the TRN neurons, whereas Li et al. 43 showed that nearly all TRN neurons expressed PV and about 64% of them expressed SST, which suggested the overlapping of STT and partly PV-expressing neurons in the TRN.To confirm the composition of TRN GABAergic neurons, we proceeded PV/SST double staining and found that about 40% of aTRN neurons are expressing both PV and SST, whereas the proportion of PV and SST neurons in aTRN is around 80% and 60% respectively (Figure S1), which is consistent with the Li's article. 43Furthermore, c-Fos staining revealed that both aTRN PV and aTRN SST neurons were activated during propofol/isoflurane induced anesthesia (Figure 1).These data indicated the involvement of both aTRN PV and aTRN SST neurons in regulation of GA.
We next generated optogenetic and chemogenetic tools to figure out the function of aTRN PV and aTRN SST neurons in GA.As predicted, optogenetic activation of both aTRN PV and aTRN SST neurons or chemogenetic activation of aTRN PV neurons promoted the induction of isoflurane anesthesia and delayed the emergence of both propofol and isoflurane anesthesia accompanied with the accumulated BSR during anesthesia.This is consistent with the former researches which showed that TRN activation induces non-rapid-eye-movement sleep (NREM) and decreases arousal. 50,51Interestingly, optogenetic or chemogenetic activation of aTRN neurons showed different effects on the power distribution of EEG frequency during anesthesia.Optogenetic activation of aTRN PV and aTRN SST neurons caused decreased δ wave (1-4 Hz) proportion and increased percentage of α wave (8-12 Hz) and γ wave (25-60 Hz) during propofol/isoflurane anesthesia, whereas δ wave (1-4 Hz) is the characteristic of EEG of GA.On the contrary, chemogenetic activation of aTRN PV neurons increased the percentage of the δ wave (1-4 Hz) and decreased the γ wave (25-60 Hz) proportion during anesthesia, which is consistent with the promotion effect of aTRN PV neurons on the induction of GA.2][53][54][55][56] TRN neurons are identified to generate spindle oscillations during sleep 32,50,51,54,56 and sustained stimulation of TRN also increases δ wave (1-4 Hz) related slow-wave sleep. 51Consequently, we observed the increased δ wave (1-4 Hz) proportion during propofol/isoflurane anesthesia after aTRN PV neuron activation by chemogenetic approaches, which may contribute to the promotion for anesthesia.In the other hand, as the pacemaker of the cortical spindles, the TRN is served as a rhythmic nucleus in the thalamus, 50,54  In our study, we did not see the different functions of aTRN PV and aTRN SST neurons in GA, although the PV and SST-expressing neurons in the TRN were reported to play different roles in many other TRN-related procedures. 48The possible reason is the overlapping of the PV and SST-expression in the aTRN neurons.Our data showed that about 40% of the aTRN neurons express both PV and SST.So the manipulation of aTRN neurons using PV-IRES-cre and SST-IRES-cre mice is hardly for us to distinguish the distinct roles of aTRN PV and aTRN SST neurons in GA.In the other hand, the PV and SST-expressing TRN neurons exhibited different electrophysiological properties and formed distinct neural circuits, 43,45,48 which indicated the distinguished mechanism of aTRN PV and aTRN SST neurons in the regulation of GA, even though they showed similar promotion effect on anesthesia induction.At last Li et al. 43 suggested that the molecular and electrophysiological properties may be a more appropriate standard for classification of the TRN neurons rather than the simply identification of PV or SST expression.
In the future, an activity dependent tool for neuron manipulation, such as the TRAP (targeted recombination in active populations), 57 may be useful to clarify the exact functions of aTRN PV and aTRN SST neurons in GA.
To further confirm the roles of aTRN GABAergic neurons in GA, we used VGAT-IRES-cre to manipulate the whole mount of the aTRN neurons.ChR2-induced activation of aTRN VGAT neurons showed comparable effects on propofol and isoflurane anesthesia to aTRN PV and aTRN SST neurons (Figure 8A-C

Furthermore
, paired Student's t-tests were used to analyze differences in the change in LORR and RORR times, the changes of EEG power bands and BSR for chemogenetic and optogenetic experiments within group (CNO vs. saline or light on vs. light off).Moreover, one-way ANOVA was used to the comparison of LORR times and RORR times and the changes of EEG power bands in optogenetic or chemogenetic experiments between groups (mCherrylight on vs. ChR2-light on or EGFP-CNO vs. M3-CNO).All data are shown as mean ± SD. p < 0.05 was considered statistically significant."n" refers the number of animals we tested in the corresponding experiments.

F I G U R E 3
Optogenetic activation of aTRN PV Neurons promotes the induction and delays the emergence of isoflurane anesthesia.(A) Schematic diagram of LORR, RORR and EEG recording during 1.4% isoflurane anesthesia.(B, C) The effect of aTRN PV neuron activation by blue laser on LORR and RORR time under 1.4% isoflurane anesthesia (LORR: mCherry-light-off: 107.63 ± 10.27 s; mCherry-light-on: 106.12 ± 10.19 s; ChR2-light-off: 114.88 ± 8.90 s; ChR2-light-on: 94.50 ± 6.61 s; RORR: mCherry-light-off: 133.25 ± 19.40 s; mCherry-lighton: 148.00 ± 25.21 s; ChR2-light-off: 146.25 ± 26.67 s; ChR2-light-on: 214.87 ± 33.45 s; n = 8).(D) The effect of TRN PV neuron activation by blue laser on BSR during the RORR period of 1.4% isoflurane anesthesia (n = 8).(E, F) The power distribution of EEG frequency bands in ChR2-light off or ChR2-light on group under 1.4% isoflurane anesthesia (n = 8).(G, H) Representative EEG wave forms and spectrograms EEG power of ChR2-light off or ChR2-light on group under 1.4% isoflurane anesthesia.Paired Student's t-tests and One-way ANOVA: (B, C).Paired Student's t-tests: (D-F).*p < 0.05, **p < 0.01.ofPV + /c-Fos + cells was observed during propofol anesthesia, whereas PV + /c-Fos + cell number was decreased after recovery of consciousness (Figure1A,B).Concurrently, similar accumulated SST + /c-Fos + expression was found in the propofol anesthesia groups compared to the wakefulness and recovery stages (Figure1C,D).These data indicate that both PV and SST neurons in aTRN are activated during propofol anesthesia.We next checked the activity of aTRN neurons during isoflurane anesthesia.Consistent with the propofol treatment, the number of both PV + /c-Fos + and SST + /c-Fos + cells in aTRN was increased during isoflurane anesthesia and decreased in the recovery period (Figure1E-H).At last, we used VGAT-td mice to confirm that the td-tomato-labeled TRN neurons are activated during both propofol and isoflurane anesthesia (Figure 1I,J).These findings suggest that both PV and SSTexpressed GABAergic neurons in aTRN may involve in the regulation of distinct stages of GA.Next, optogenetic and chemogenetic tools are used to specifically manipulate the activity of PV or SST-expressed aTRN GABAergic neurons (aTRN PV or aTRN SST ) to clarify its precise function in GA.F I G U R E 4 Optogenetic activation of aTRN SST Neurons decelerates the emergence of propofol anesthesia.(A) Representative images of optogenetic virus (ChR2-mCherry, red) expression in aTRN SST neurons (green, scale bar, 50 μm).(B) Ex vivo electrophysiology of ChR2 virus action in aTRN SST neurons.(C) The effect of aTRN SST neuron activation by blue laser on RORR time under propofol anesthesia (mCherrylight-off: 307.00 ± 30.87 s; mCherry-light-on: 313.25 ± 22.38 s; ChR2-light-off: 286.88 ± 23.91 s; ChR2-light-on: 378.25 ± 24.62 s; n = 8).(D) The effect of aTRN SST neuron activation by blue laser on BSR during the RORR period of propofol anesthesia (n = 8).(E) The power distribution of EEG frequency bands in ChR2-light off or ChR2-light on group under propofol anesthesia (n = 8).(F, G) Representative EEG wave forms and spectrograms EEG power of ChR2-light off or ChR2-light on group under propofol anesthesia.Paired Student's t-tests and One-way ANOVA: (C).Paired Student's t-tests: (D, E). *p < 0.05, **p < 0.01.

Furthermore,
EEG recordings showed a higher δ wave (1-4 Hz) proportion and a lower percentage of γ wave (25-60 Hz) during the whole progression of isoflurane anesthesia in CNO treated M3-expressing mice, including induction, maintenance and emergence period (Figure 7E-I, Figure S4D-I).These results indicate that continuous activation of aTRN PV neurons accelerates isoflurane induction and delays the recovery of both propofol and isoflurane anesthesia.
indicated possible distinct functions of different subtypes of TRN GABAergic neurons.However, inconsistent results have been reported in the previous studies.Clemente-Perez et al. 48reported that PV-and SST-expressing cells are two distinct distinguished subtypes | 13 of 17 YI et al.
so blue-light stimulation with distinct frequency on aTRN PV or aTRN SST neurons may generate the cortical EEG band in specific frequency.In our optogenetic experiments, 10 Hz bluelight stimulation on both aTRN PV and aTRN SST neurons caused massive accumulation of 10 Hz EEG band (Figures 2H, 3H, 4G and 5G), which resulted in the increased percentage of α wave (8-12 Hz).The decreased δ wave (1-4 Hz) proportion may be the compensation in response to α wave increase.Besides the different effects on EEG composition, optogenetic and chemogenetic activation of aTRN neurons suggested the shared roles of aTRN PV and aTRN SST neurons in regulation of propofol/isoflurane anesthesia.

| 15 of 17 YI
), which suggested the shared mechanism of different subtypes of aTRN GABAergic neurons in GA.Interestingly, M4-mediated inhibition of aTRN VGAT neurons only showed influence on the induction of isoflurane anesthesia (Figure8F), but not the recovery of propofol or isoflurane anesthesia (Figure8E,F).This may be attributed to the accumulated activity of TRN neurons during anesthesia and the activity recovery during the emergency period.In the ChR2 experiment, continuous activation of aTRNVGAT  neurons during the RORR stage sustained the maintenance of anesthesia to delay the recovery.On the contrary, F I G U R E 6 Chemogenetic activation of aTRN PV Neurons delays the emergence of propofol anesthesia.(A) Up: schematic diagram of virus injection into aTRN and EEG electrode implantation.Down: schematic diagram of RORR and EEG recording during propofol anesthesia by single dose of 20 mg/kg injection.(B) Representative images of chemogenetic virus (M3-EGFP, green) expression in aTRN PV neurons (red, scale bar, 50 μm).(C) Ex vivo electrophysiology of M3 virus action in aTRN PV neurons.(D) The effect of aTRN PV neuron activation by CNO on RORR time under propofol anesthesia (EGFP-saline: 290.13 ± 22.20 s; EGFP-CNO: 286.38 ± 22.60 s; M3-saline: 275.00 ± 26.07 s; M3-CNO: 360.38 ± 40.53 s; n = 8).(E) The effect of aTRN PV neuron activation by CNO on BSR during the RORR period of propofol anesthesia (n = 8).(F) The power distribution of EEG frequency bands in M3-saline or M3-CNO group under propofol anesthesia (n = 8).(G, H) Representative EEG wave forms and spectrograms EEG power of M3-saline or M3-CNO group under propofol anesthesia.Paired Student's t-tests and One-way ANOVA: (D).Paired Student's t-tests: (E, F). *p < 0.05, **p < 0.01.et al.M4-mediated inhibition of aTRN VGAT neurons blunted the activation of aTRN neurons, which is critical for the induction and maintenance of anesthesia.Taken together, our data suggest that aTRN GABAergic neurons may involve in the regulation of distinct stages of GA.AUTH O R CO NTR I B UTI O N S All authors contributed to the experimental conception and design of the study.Material preparation, data collection, and analyses were performed by R.Y., S.C., F.Z., Y.Y., L.Z. and Y.Z.R.Y., S.C. and D.L. generated all the mouse models.The manuscript was written by F I G U R E 7 Chemogenetic activation of aTRN PV neurons promotes the induction and delays the emergence of isoflurane anesthesia.(A) Schematic diagram of LORR, RORR and EEG recording during 1.4% isoflurane anesthesia.(B, C) The effect of aTRN PV neuron activation by CNO on LORR and RORR time under 1.4% isoflurane anesthesia (LORR:EGFP-saline: 109.63 ± 9.35 s; EGFP-CNO: 109.63 ± 8.30s; M3-saline: 122.37 ± 8.98 s; M3-CNO: 94.25 ± 7.38 s; RORR:EGFP-saline: 131.13 ± 12.39 s; EGFP-CNO: 146.37 ± 19.05 s; M3-saline: 146.37 ± 15.11 s; M3-CNO: 230.75 ± 25.79 s; n = 8).(D) The effect of aTRN PV neuron activation by CNO on BSR during 1.4% isoflurane anesthesia (n = 8).(E-G) The power distribution of EEG frequency bands in M3-saline or M3-CNO group under 1.4% isoflurane anesthesia (n = 8).(H, I) Representative EEG wave forms and spectrograms EEG power of M3-saline or M3-CNO group under 1.4% isoflurane anesthesia.Paired Student's t-tests and One-way ANOVA: (B, C).Paired Student's t-tests: (D-G).*p < 0.05, **p < 0.01.F I G U R E 8 Manipulation of aTRN VGAT neurons regulates the behavior of propofol and isoflurane anesthesia.(A) Representative images of optogenetic virus (ChR2-EGFP, green) expression in TRN VGAT neurons (red, scale bar, 50 μm).(B) The effect of aTRN VGAT neuron activation by blue laser on RORR times under propofol anesthesia (n = 8).(C) The effect of aTRN VGAT neuron activation by blue laser on LORR and RORR times under 1.4% isoflurane anesthesia (n = 8).(D) Representative images of chemogenetic virus (M4-EGFP, green) expression in aTRN VGAT neurons (red, scale bar, 50 μm).(E) The effect of aTRN VGAT neuron inhibition by CNO on RORR times under propofol anesthesia (n = 8).(F) The effect of aTRN VGAT neuron inhibition by CNO on LORR and RORR times under 1.4% isoflurane anesthesia (n = 8).Paired Student's t-tests: (B, C, E, F). *p < 0.05, **p < 0.01.